The long-term goals of this project are to develop methods to probe the organization and dynamic reorganization of biological membranes. This includes interactions among the components within membranes, interactions between membrane surfaces that lead to binding, fusion, and pattern formation, and conformational changes of proteins associated with membranes. The lipid bilayer is the basic structure common to biological membranes. Membrane fluidity is critical for biological functions that depend upon conformational changes within membranes, the lateral association or clustering of multiple components, and processes that change membrane topology such as edo- and exocytocis and fusion. This proposal outlines new types of experiments that probe these basic aspects of membrane dynamics using tools that have been developed to pattern, manipulate and image supported bilayers. During the next grant period the focus will be on the mechanism of vesicle fusion, using vesicles that are tethered to supported bilayers and whose interactions can be monitored at the level of individual vesicles (Aim 1);the lateral association and organization of lipids and membrane anchored proteins using a novel type of imaging mass spectrometry that permits membrane composition analysis with unprecedented lateral resolution, sensitivity and information content (Aim 2);and the design and fabrication of an integrated optical/electrical device that will permit high precision interferometry on planar bilayers to probe conformational transitions of membrane-associated proteins, with an initial focus on voltage-gated ion channels (Aim 3). Each aim depends upon the development of new supported lipid bilayer architectures and analytical methods that can have a broad impact on studies of biological membranes. Relevance to human health: A significant fraction of all proteins are associated with membranes, and, as a class, these constitute a huge and diverse target for drug development. This proposal outlines new methods for studying membranes and membrane-associated proteins that can impact our understanding of biological function and organization, as well as impact biotechnology.